Trehalose [α-D-glucopyranosyl α-D-glucopyranoside] is a naturally occurring nonreducing disaccharide in which the two glucose molecules are linked through an 1,1-glycosidic bond. Though three anomers of trehalose exist, which are α,α-1,1-, α,β-1,1,- and β,β-1,1-trehalose, only the α,α-1,1-form is widespread in nature and present in a large number of organisms, including bacteria, yeast, fungi, insects, invertebrates, and plants (1). The function of trehalose in organisms varies. In addition to serving as an energy and carbon source, trehalose is the basic component of various cell wall glycolipids of mycobacteria and corynebacteria, which adds to the impermeability of the cell walls and has been implicated in the pathogenesis of diseases caused by such bacteria (2, 3).
In yeast and plants, trehalose or a related metabolite, trehalose-6-phosphate, acts as a signaling or regulatory molecule that interferes with pathways associated with energy metabolism or even affects growth and development (4-6). Moreover, trehalose has been found to help organisms acquire tolerance to various stresses, including cold, heat, desiccation, dehydration, and osmotic and oxidative stress (1, 7-10). This quality is attributed to its “chemical chaperon” property, namely its ability to maintain proteins and membranes in their native conformations (1, 8, 11), or to its presumed ability to quench the oxygen radical (10).
Three main pathways specifying the biosynthesis of trehalose have been identified in various organisms (12). The first pathway utilizes trehalose-phosphate (P) synthase (EC 2.4.1.15) (OtsA in Escherichia coli) that catalyzes the transfer of glucose from UDP-glucose to glucose-6-P to form trehalose-P and UDP. The phosphate is then removed by trehalose-P phosphatase (EC 3.1.3.12) (OtsB in E. coli) to give free trehalose (13).
The second pathway also involves two enzymes called maltooligosyl trehalose synthase (EC 5.4.99.15) and maltooligosyl trehalose trehalohydrolase (EC 3.2.1.141). The former enzyme first converts the α1,4-linkage in the reducing end of the maltooligosaccharide chain into the α1,1-linkage and then the latter enzyme hydrolyzes the reducing-end disaccharide to release one molecule of trehalose (14-16).
The third pathway, catalyzed by trehalose synthase (TSase) (EC 5.4.99.16), involves the direct conversion of maltose into trehalose by an intramolecular rearrangement of the α-1,4-linkage of maltose to the α-1,1-linkage of trehalose (17). Since this pathway allows one-step formation of trehalose and an inexpensive substrate, maltose, is employed, it is highly useful for the industrial manufacture of trehalose.
At this time, approximately six different trehalose synthases have been reported from different species and characterized for their biochemical properties (18-23). However, the trehalose synthases of the prior art are characterized by biochemical properties, including their low tolerance for heat and acidity, that may be undesirable in certain applications or in use in certain environments. For example, the three trehalose synthases from Pimelobacter sp. R48, Thermobifida fusca, and Pseudomonas stutzeri CJ38 are thermolabile. Although the trehalose synthase from Thermus aquaticus on the contrary is highly thermostable, this enzyme has the drawback of low enzyme yield in the original organism, which is undesirable for industrial scale preparation of the enzyme because a culture of a large volume of organisms is required for enzyme isolation. In addition, the trehalose synthase enzymes are most active at a pH that is near or above neutral, which precludes these enzymes from being useful in even slightly acidic environments. Therefore, there is a current need in the art for a trehalose synthase enzyme which retains high degree of activity at higher temperatures and in more acidic environments than the trehalose synthases of the prior art.
The present invention teaches the biochemical properties of a novel recombinant trehalose synthase from Picrophilus torridus, a hyperacidophilic, thermophilic, heterotrophic and absolutely aerobic archaea, which grows optimally at 60° C. and pH 0.7 (13). The novel Picrophilus torridus trehalose synthase of the present invention has superior heat resistant and acid resistant qualities when compared to trehalose synthases of the prior art. The heat resistant and acid resistant trehalose synthase of the present invention offers a significant advantage for industrial production of trehalose by increasing the solubility of substrate and reducing the risk of contamination during production.